Synthesis of N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units for the assembly of backbone cyclic peptides.

To improve the assembly of backbone cyclic peptides, N-functionalized dipeptide building units were synthesized. The corresponding N-aminoalkyl or N-carboxyalkyl amino acids were formed by alkylation or reductive alkylation of amino acid benzyl or tert-butyl esters. In the case of N-aminoalkyl amino acid derivatives the aldehydes for reductive alkylation were obtained from N,O-dimethyl hydroxamates of N-protected amino acids by reduction with LiAlH4. N-carboxymethyl amino acids were synthesized by alkylation using bromoacetic acid ester and the N-carboxyethyl amino acids via reductive alkylation using aldehydes derived from formyl Meldrums acid. Removal of the carboxy protecting group leads to free N-alkyl amino acids of very low solubility in organic solvents, allowing efficient purification by extraction of the crude product. These N-alkyl amino acids were converted to their tetramethylsilane-esters by silylation with N,O-bis-(trimethylsilyl)acetamide and could thus be used for the coupling with Fmoc-protected amino acid chlorides or fluorides. To avoid racemization the tert-butyl esters of N-alkyl amino acids were coupled with the Fmoc-amino acid halides in the presence of the weak base collidine. Both the N-aminoalkyl and N-carboxyalkyl functionalized dipeptide building units could be obtained in good yield and purity. For peptide assembly on the solid support, the allyl type protection of the branching moiety turned out to be most suitable. The Fmoc-protected N-functionalized dipeptide units can be used like any amino acid derivative under the standard conditions for Fmoc-solid phase synthesis.     

Olfaction in peking ducks (Anas platyrhynchos): A comparative study of centrifugal and centripetal olfactory connections in young ducks and in embryos and ducklings (Aves)

Summary Our study of young white Peking ducks (Anas platyrhynchos) revealed that their main olfactory centres are very similar to those reported for pigeons. Additionally, two major olfactory tracts and other olfactory centres were found to be intimately associated with telencephalic and diencephalic limbic structures. Our results make it possible to reject the view that olfaction in birds is not connected with the limbic system, as is the case in other vertebrates. The occurrence of olfaction was found to be considerably higher in embryos and ducklings. Embryonic connections are more extensive than the olfactory structures of young ducks, covering the archipallial wall of the hemisphere, i.e. the dorsomedial hippocampus and the main laminae of the forebrain. Transitory centripetal olfactory connections develop in embryos, and centrifugal olfactory connections are present in ducklings at the time of hatching. Controls revealed that a portion of the olfactory connections is drastically reduced by physiological degeneration involving connections emanating from both directions. Ultrastructural examination of embryonic archipallial structures revealed that the degeneration is partially the result of the dispersal of innervated neurons. Phylogenetically, one may compare the significant involvement of transitory olfactory connections in the anlage of limbic structures with the conditions occurring in anamniot vertebrates. This supports the hypothesis that the primary development of transitory olfactory connections represents an ontogenetic recapitulation of ancestral conditions, by which the structural and functional organization of the avian brain is influenced.     

DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.     

The cytoskeletal architecture of interdigitated microvilli in the photoreceptors of some nocturnal spiders

Summary The photoreceptor microvilli of some nocturnal spiders (Isopeda andOlios in theSparassidae, andClubiona in theClubionidae) are wide (80–140 nm), and microvilli from adjacent receptors are interdigitated. Because microvillar diameters are relatively large in relation to the thicknesses of thin sections, it is possible to examine cytoskeletal structures closely associated with the microvillar plasmalemmae directly.Retinae were treated with a specific inhibitor of cysteine proteases before primary fixation for electron microscopy in a Ca²⁺-chelating medium. Cytoskeletal components were stabilized with tannic acid. A variety of microvillar profiles was obtained, consistent with an assumption that they represent imperfect preservation of anin vivo plasmalemmal undercoat, inferred to consist of longitudinally-disposed microfilaments, presumptively bonded to the microvillar plasmalemma. The microvillar lumen is inferred to be empty of cytoskeletal components in life.This model is discussed in terms of 1. the cytoskeletal organisation of microvilli of the primitive photoreceptors of a leech (Blest et al. 1983), where the arrangement of microfilaments resembles that in the vertebrate intestinal brush-border; 2. the large complement of membrane-associated oligomeric actin in rhabdoms of crayfish, where identifiable microfilaments cannot be resolved within microvilli by transmission electron microscopy (de Couet et al. 1984), and a single visualizable axial filament of uncertain composition is linked to the plasmalemma by side-arms.     

Spectinomycin resistance due to a mutation in an rRNA operon of Escherichia coli

A spectinomycin resistance mutation was isolated in an Escherichia coli rRNA operon (rrnH) located on a multicopy plasmid. Cell-free protein-synthesizing extracts made from cells containing the plasmid were partially resistant to spectinomycin. Although spectinomycin is an aminoglycoside antibiotic, the mutation did not confer resistance to any other aminoglycoside antibiotic tested.     

EFFECT OF ERYTHROPOIETIN ON PROLIFERATION OF ERYTHROPOIETIN-RESPONSIVE CELLS

A single dose of Myleran suppressed CFU in polycythemic mice to around 1% of normal for a period of 2 weeks and permitted the study of effects of erythropoietin on unipotential, erythroid stem cells (erythropoietin-responsive cells, ERC) in the absence of cell inflow from the CFU compartment. Without erythropoietin no ERC were detectable for 12 days after Myleran. Injections of erythropoietin had no effect on CFU but restored ERC populations in proportion to the dose of erythropoietin. Hydroxyurea given after erythropoietin markedly inhibited ERC repopulation and the latter is attributed to a stimulation of ERC proliferation by erythropoietin. Evidence in support of an age structure in the ERC population is presented. Daily erythropoietin injection resulted in stable ERC populations, indicating that ERC loss through differentiation and ERC self-replication were in balance.     

Augmentation of host antitumor immunity by low doses of cyclophosphamide and mafosfamide in two animal tumor models

Summary By cloning in vitro we have obtained two sublines of the L5222 rat leukemia, one with high (L5222-S) and the other with low (L5222-R) in vivo sensitivities to non-toxic doses of mafosfamide, a stabilized derivative of 4-hydroxy-cyclophosphamide. This sensitivity in vivo was not related to the cytotoxic activity of the drug in vitro. Treatment of rats bearing the L5222-S and of mice transplanted with the MOPC-315 plasmocytoma with low doses of mafosfamide or cyclophosphamide resulted in a high percentage of surviving animals, which were resistant to a subsequent tumor challenge. Viable leukemic cells were needed to establish antitumor immunity, since it was not possible to induce resistance by injection of mitomycin-C-treated, non-viable L5222 cells. The adoptive transfer of spleen cells from animals immune against the L5222-S and the MOPC-315 resulted in resistance of the syngeneic recipients against a rechallenge with tumor cells, provided that the animals were treated with an immunosuppressive dose (100 mg/kg) of cyclophosphamide prior to the spleen cell implantation. In nude mice treatment of the L5222 with low doses of mafosfamide also resulted in surviving animals, however resistance to a second tumor challenge occurred only sporadically.The data presented confirm that therapy with cyclophosphamide or mafosfamide enhances host antitumor immunity but, contrary to previous reports, it could be demonstrated that successful tumor rejection was independent of T cells.Supported by the Federal Ministry of Research and Technology (BMFT), Bonn-Bad Godesberg, FRG     

Induction of histamine release from rat mast cells by bradykinin analogues

Independently of their agonistic or antagonistic activity on different isolated tissue preparations, the kinin analogues investigated induce histamine release on rat peritoneal mast cells. The effectivity of most compounds is 10 to 100 times higher than that of bradykinin. Beside the positively charged amino acids, the elongation at the N-terminus with hydrophobic amino acids and the replacement of amino acids in the bradykinin sequence (especially at position 7) with aromatic residues is important for a high histamine-releasing activity.